Recent advances in tissue culture techniques have established the potential for commercialization of large-scale proliferation for numerous plant species. For rice (Oryza sativa L.), techniques which suggest the possibility of large-scale proliferation have been reported. However, it is unclear whether they have attained a commercially applicable level of efficiency.
Two techniques have been proposed for proliferating rice by tissue culture. The first involves differentiating and inducing the formation of an adventitious bud or an somatic embryo directly from an explant tissue to proliferate rice plants. This technique has been reported (Ling, D. H. et al., Plant Cell Reports 2, 172 (1983); Wernicke, W. et al., Z. Pflanzenphysiol. 103, 361 (1981); Eur. J. Cell Biol. 24, 347 (1981); Stuart, D. A. and S. G. Strickland, WO 87/02701). In this technique, however, the differentiation and induction ratios reported for individuals or somatic embryos are very low. Thus, this technique appears impracticable from the standpoint of proliferation efficiency for large-scale proliferation on a commercial basis.
A second technique involves inducing and proliferating a callus culture from a rice explant tissue, then inducing and producing a somatic embryo or an adventitious bud from the callus tissue and finally regenerating the embryo or adventitious bud into a whole plant. Although many sites on a rice plant, such as ovary, anther, immature embryo, full-ripe seed, juvenile leaf, root, shoot apex, juvenile panicle, etc., can be used as explants for the induction of rice callus, the reported redifferentiation ratios are low. Thus, this technique has not been shown to be practical as a proliferation technique for obtaining a large number of regenerated individual plants from a small amount of explant material.
As pointed out by Nabors, W. M. and J. W. Heyser (Plants 157., 385 (1983)), Siriwardana, S. and M. W. Nabors (Plant Physiol. 73, 143 (1983)), and others, calli induced from explant material can be classified into two groups, that is, embryogenic callus having embryogenic potency (hereinafter referred to as "E callus") and nonembryogenic callus having no embryogenic potency (hereinafter referred to as "NE callus"). In many cases, both types of calli are present as a mixture in culture. It has been found that the individual regeneration potency of the overall callus culture is directly related to the proportion of E callus present. However, the proportion of NE callus in a culture generally increases with the passage of time and as proliferation progresses, and thus the individual regeneration potency tends to decrease. It is believed that this phenomenon is due to a higher proliferation rate for NE callus than E callus, and because E callus can convert to NE callus in culture, whereas NE callus will not convert to E callus.
In order to improve the redifferentiation ratio of rice tissue culture, it may be possible, e.g. to induce E calli from explants and serially transfer the E calli to allow proliferation without conversion to NE calli; to separate E calli from the induced calli; to invent a selective medium in which E calli alone can be proliferated; or the like. However, there are no known reports of practicable techniques based on these ideas.
In order to make large-scale proliferation a commercially applicable technique, it is necessary to induce and proliferate a suspension culture consisting of E calli alone. Generally, a suspension culture is obtained by placing an explant on an agar medium containing auxin, inducing a callus and then transferring the callus into a liquid medium. Examples of the culture of an explant performed in a liquid medium from the beginning of culture include the reports by Zimny and Lorz (Plant Cell Reports 5, 89 (1986)) and Toriyama and Hinata (J. Breed. 36, suppl. 2, 50 (1986)) among others. However, in these examples, NE calli constituted the majority of the suspensions obtained. The redifferentiation ratio obtained by Toriyama and Hinata (1986) was as low as 2% per callus.
P. B. Kavikishor (Plant Science 48, 189 (1987)) suggested that the addition of an osmotic regulator to an agar medium is effective for the proliferation of callus and the redifferentiation of foliage. In this report, however, the redifferentiation ratio stayed at a low frequency of 1 individual plant per 300 mg of callus. Stuart and Strickland (supra, (1987)) suggested that the addition of various amino acids, particularly L-proline, was effective for the formation of somatic embryos from callus. In addition, other reports have suggested that casein hydrolysate, yeast extract, etc., are effective substances for increasing redifferentiation efficiency (Abe and Futsuhara [Japan J. Breed. 34, 147 (1984); Japan Physiol. 121, 111 (1985) and Japan J. Breed. 36, 1 (1986)]; Ling, D. H. et al., Plant Cell Reports 2, 169 (1983)). In these reports, the respective substances were said to promote a maximum redifferentiation efficiency of 300 individuals/g callus by their independent use. In the report of Raghavaram, N. V. and M. W. Nabors (Plant Cell Tiss. Organ Cult. 4, 241 (1985)), a redifferentiation ratio near 300 individuals/g callus was obtained by using a scutellum-derived callus. However, these reports were made relating to particular cultivars such as Chyokoto, Pokkali, Taipei 309, etc. and the application of their techniques to ordinary cultivars did not produce such redifferentiation ratios.